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Grain protein content (GPC) in wheat has been found to be regulated by the gene NAM-B1. Homologues to the NAM-B1 gene have been found in barley, HvNAM-1 and HvNAM-2. Previous studies have found that base mutations in the NAM-1 gene at base position 544 might have an impact on GPC. Previous studies also found that landrace of barley showed higher GPC than cultivated barley, indicating that plant improvement might have affected base mutations and therefore GPC. I wanted to study if there are any nutritional differences in Nordic barley and if those differences might correlate with haplotypes. Comparisons of barley varieties from four Nordic countries, and two varieties from the US used as low and high GPC controls, did not show any significant differences depending on their origin country and no differences regarding plant improvement status between the countries. When sequencing Nordic barley varieties, five haplotypes were found for the gene HvNAM-1, and two haplotypes for the gene HvNAM-2. A low polymorphism for both genes indicate a strong natural selection for the consensus haplotype which might be preferable for Nordic climate with a short growing season and cold temperatures. Even though it is not clear what is the cause of the low polymorphism in Nordic barley varieties, they showed a generally higher nutrient content than barley varieties of the high GPC and may be suitable for breeding for a yield with a high nutrient content.

Several yeast species had previously been isolated from water systems in the North West Province, South Africa. Some of the identified species had, in other studies, been associated with superficial mucosal infections to life threatening diseases. Antifungal drugs are used to treat such yeast infections. However, due to prophylactic usage and continuous exposure some yeast species have developed resistance to some antifungal agents. The aim of this study was to determine the diversity and antifungal susceptibility of yeasts in selected rivers, Mooi River and Harts River in the North West Province, South Africa. Waters samples were collected from the rivers in summer and winter seasons. Physico-chemical parameters such as pH, temperature, total dissolved solids, chemical oxygen demand, nitrates and phosphates were measured to determine the water quality. Yeast colonies were enumerated at room temperature and 37°C using yeast-malt-extract agar (containing 100 ppm chloramphenicol). Pure isolates from 37°C were identified by biochemical tests and 26S rRNA gene sequencing. Yeast sequences of isolated yeasts were sent to Genbank. Phylogenetic tree was conducted to determine phylogenetic relationship between the yeast isolates. Disk diffusion antifungal susceptibility tests were conducted on the yeast species. Physico-chemical parameters of the water were within target water quality range for livestock farming but in most sampling sites out of range for irrigation use. pH, Nitrates, phosphates and chemical oxygen demand levels ranged from 7.40 to 8.64, 0 to 5.4 mg/L, 0 to 7.14 mg/L and 31 to 43 mg/L, respectively. Elevated levels of total dissolved solids were measured in all the sampling sites. Total yeast counts ranged between 320-4200 cfu/L and 27-2573 cfu/L for room temperature and 37˚C. All the yeast colonies isolated were non-pigmented. Diazonium Blue B tests determined the yeasts isolates as ascomycetes. Haemolysin and extracellular enzyme production tests were negative on all the isolates. Yeasts isolates were identified and belonged to the genera Arxiozyma, Candida, Clavispora, Cyberlindnera, Lecythophora, Pichia, Saccharomyces, and Wickerhamomyces. Saccharomyces cerevisiae and Candida glabrata were mostly isolated species. Furthermore, the results indicated that levels of yeast could be correlated to physico-chemical quality of water. A large number of isolates were resistant to azoles, especially fluconazole as well as other antifungal classes. Most of the Candida species were resistant to almost all the antifungals. Several of the isolated yeast species are opportunistic pathogens. They could cause infections in sensitive individuals during occasional direct contact especially immune compromised people. Resistance of these yeast species to antifungal agents is a major health concern. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Several yeast species had previously been isolated from water systems in the North West Province, South Africa. Some of the identified species had, in other studies, been associated with superficial mucosal infections to life threatening diseases. Antifungal drugs are used to treat such yeast infections. However, due to prophylactic usage and continuous exposure some yeast species have developed resistance to some antifungal agents. The aim of this study was to determine the diversity and antifungal susceptibility of yeasts in selected rivers, Mooi River and Harts River in the North West Province, South Africa. Waters samples were collected from the rivers in summer and winter seasons. Physico-chemical parameters such as pH, temperature, total dissolved solids, chemical oxygen demand, nitrates and phosphates were measured to determine the water quality. Yeast colonies were enumerated at room temperature and 37°C using yeast-malt-extract agar (containing 100 ppm chloramphenicol). Pure isolates from 37°C were identified by biochemical tests and 26S rRNA gene sequencing. Yeast sequences of isolated yeasts were sent to Genbank. Phylogenetic tree was conducted to determine phylogenetic relationship between the yeast isolates. Disk diffusion antifungal susceptibility tests were conducted on the yeast species. Physico-chemical parameters of the water were within target water quality range for livestock farming but in most sampling sites out of range for irrigation use. pH, Nitrates, phosphates and chemical oxygen demand levels ranged from 7.40 to 8.64, 0 to 5.4 mg/L, 0 to 7.14 mg/L and 31 to 43 mg/L, respectively. Elevated levels of total dissolved solids were measured in all the sampling sites. Total yeast counts ranged between 320-4200 cfu/L and 27-2573 cfu/L for room temperature and 37˚C. All the yeast colonies isolated were non-pigmented. Diazonium Blue B tests determined the yeasts isolates as ascomycetes. Haemolysin and extracellular enzyme production tests were negative on all the isolates. Yeasts isolates were identified and belonged to the genera Arxiozyma, Candida, Clavispora, Cyberlindnera, Lecythophora, Pichia, Saccharomyces, and Wickerhamomyces. Saccharomyces cerevisiae and Candida glabrata were mostly isolated species. Furthermore, the results indicated that levels of yeast could be correlated to physico-chemical quality of water. A large number of isolates were resistant to azoles, especially fluconazole as well as other antifungal classes. Most of the Candida species were resistant to almost all the antifungals. Several of the isolated yeast species are opportunistic pathogens. They could cause infections in sensitive individuals during occasional direct contact especially immune compromised people. Resistance of these yeast species to antifungal agents is a major health concern. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

In eukaryotic cells, genomic DNA exists as chromatin in association with histone octamers called nucleosomes, and various other chromatin proteins. Chromatin structure varies along the chromosome and this influences the state of gene expression. Based on such variations in structure and gene expression, chromatin can be broadly classified into euchromatin (transcriptionally active) and heterochromatin (silent or transcriptionally repressed). In the budding yeast, Saccharomyces cerevisiae, there are four canonical transcriptionally silent regions, namely, the HMR, the HML (cryptic mating loci), the telomeres and the RDN1. Silencing at the HM loci and the telomeres is very well characterized. The repressive structure at the HMR spans around 3.5 Kb and extends between the two silencers E and I. It is well established that silencing in HMR is due to a specialized chromatin organization brought about by Orc1p, Rap1p, Abf1p and Sir proteins. Following recruitment, the Sir proteins spread along the DNA to form a repressive chromatin domain believed to arise from the deacetylation of amino-terminal tails of histones H3 and H4 by Sir2p (an NAD dependent deacetylase) and the interaction of Sir3p and Sir4p with the histones. The bi-directional spreading of silencing at HMR is restricted by barrier or boundary elements that flank the silencers. A tRNAThr gene in the right boundary of HMR acts as a strong barrier. Mutations in the promoter of this tRNA gene (tDNA) or in RNA polymerase III subunits/ transcription factors weaken the barrier activity of this tDNA. The barrier activity of this tDNA is also dependent on histone acetyltransferases like Sas2p and Gcn5p. Silencing in HML is uniformly high between the silencers E and I and falls sharply outside I. Recently, barriers to HML silencing have been discovered. A 0.71Kb sequence near E, which maps to the upstream activating sequence of YCL069W, acts as a robust barrier to spread of HML silencing. This is effectively the left boundary of silent HML. The right boundary maps to the promoter of CHA1 gene though silencing is believed to terminate at HML-I. An unusual form of silencing occurs at the RDN1, which contains 100-150 copies of tandemly repeated rRNA genes. Some RNA polymerase II transcribed genes integrated within the array are silenced by a Sir2p dependent mechanism whereas genes driven by RNA polymerases III and I are transcriptionally active. Though all the three forms of silencing (RDN1, HM and telomere) require Sir2p, RDN1 silencing differs from the others in its relative strength and factors responsible for repression. Several trans-acting factors required for RDN1 silencing are known. However, it is still unclear as to what limits the spread of RDN1 heterochromatin into neighbouring essential genes. RDN1 silencing spreads unidirectionally in its left hand side sequence. However, the zone of RDN1 heterochromatin does not engulf the essential gene, ACS2, which is present ~3 kb away from NTS1. This implies that there is a mechanism by which rDNA heterochromatin is contained. There could be several ways by which this is accomplished. Firstly, the cell could be critically maintaining the levels of Sir2p, the protein required for silencing at all the four silenced loci, such that silencing in the left flank of RDN1 does not spread beyond 300 bp of NTS1 (Buck et al, 2002). There is a ~2.5 kb gene free intervening sequence between NTS1 of the rDNA array and the Ty1 LTR, in which interval Sir2p level could fall below the threshold mark required for causing repression. In fact Buck et al. have demonstrated that Sir2p is bound to upto 1.5 kb from the NTS1 in the left flank but there is no accompanying silencing of the mURA3 reporter in these regions (1200L and 2000L), suggesting that the level of Sir2p at these sequences could be lower than the threshold required for initiation of silencing. Secondly, there could be cis-acting boundary elements or barriers as in the case of HMR, which prevents the propagation of RDN1 silencing. The third option is that termination of RNA polymerase I transcription at the terminator sites automatically halts the spread of rDNA silencing since Buck et al. have demonstrated that progression of rDNA heterochromatin is dependent on RNA pol I transcription. This however, does not seem to be the case as deletion of both the terminator sites within NTS1 does not lengthen the zone of silencing. Finally, there could be an euchromatin organizing center further from the array, which creates an “open” chromatin configuration required to confront the Sir2p mediated condensed chromatin. The balance of these two opposing activities, much like that at the telomeres, could set up a molecular boundary for containing rDNA silent chromatin. We have attempted to identify whether there are any sequences in the unique left flank of RDN1 that can act as a heterochromatin barrier. Towards that end we tested four overlapping fragments from NTS1 of RDN1 to the promoter of ACS2 for boundary activity in a quantitative mating assay. We have found that of all the four fragments tested, only a 0.427 kb tRNAGln-Ty1 LTR fragment, which is present 2.4 Kb from the NTS1 acts as a robust barrier in this assay. Further mapping revealed that the barrier activity of this sequence resides in the tRNAGln gene and that its activity is orientation-independent. tDNAs are transcribed by RNA polymerase III from internal promoters termed Box A and Box B. It has been shown for the HMR-tRNAThr that the transcriptional potential of the tDNA is crucial for its barrier function. Mutations in genes encoding various subunits of the RNA polymerase III complex, or transitions in the conserved bases within Box B known to disrupt transcription complex assembly and subsequent transcription, abrogate the barrier activity of HMR-tRNAThr. Similarly, loss of transcriptional ability of the tRNAAla in the centromere of S. pombe also abolishes its barrier activity, enforcing the fact that RNA polymerase III transcription is a decisive factor for a tDNA barrier. Contrary to the above observations, we report that barrier activity of tRNAGln is very negligibly dependent on RNA polymerase III mediated transcription. Mating assays done with the RNA pol III mutants and promoter point mutants, G18C and C55G in boxes A and B respectively, underline the fact that for this tDNA barrier, RNA pol III driven transcription is dispensable. We also show by RT-PCR analysis that in the C55G tRNAGln mutant there is loss of transcription as expected, whereas other wild type copies of tRNAGln are transcribed. Studies with another tDNA barrier, TRT2-tRNAThr, yielded similar results, again emphasizing the point that transcription through the tDNA, which leads to nucleosome displacement and therefore barrier activity, may not be applicable for all tDNA barriers. Acetylation of amino terminal tails of histones is known to influence the epigenetic state of chromatin. Addition of acetyl moiety to histones H3 and H4 initiates a cascade of events, which involves recruitment of a host of other chromatin modifiers to the target sequence, and ultimately culminates in the formation of an euchromatin-favouring environment. As reported for the HMR right boundary, we find that barrier activity of tRNAGln depends on two histone acetyl transferase complexes, SAS-I (comprised of Sas2p, Sas4p and Sas5p) and SAGA (contains Gcn5p HAT). Contrary to the HMR boundary, the barrier activity of tRNAGln is independent of two other nucleoplasmic HATs, NuA3 (Sas3p being the HAT) and NuA4 (Esa1p is the HAT). Barrier function of TRT2-tRNAThr also depends on HATs. Therefore it appears that requirement of HATs for boundary activity is a conserved theme, albeit with differential effects at different barrier sequences. We next attempted to determine the function of tRNAGln in its natural location on chromosome XII. As mentioned earlier, RDN1 silencing spreads upto ~0.3 kb in its left flanking sequence. However, Sir2p occupancy has been observed till 1.5 kb although there is no silencing of reporter genes observed beyond 0.3 kb of NTS1. This lead us to speculate that there could be a boundary sequence in the left flank that stops silencing, or a euchromatin-organizing element, which counters the propagation of silencing by a long-range effect. Since over expression of Sir2p extends the domain of silencing from 0.3 kb to 2.0 kb and the tRNAGln is present at 2.3 kb from NTS1, it was a good candidate for a heterochromatin barrier/ euchromatiniser. However, deletion of tRNAGln does not affect the zone of RDN1 silencing as is evident from our cell viability assays (which is a measure of the expression of the essential gene ACS2 situated further to the left of tRNAGln). Deletion of SAS2 and GCN5, factors that are required for barrier activity of tRNAGln in mating assays, also have no effect on the extent of spreading of RDN1 silencing in normal or Sir2p over expression conditions. Together, these observations imply that in situ, tRNAGln does not act as a barrier or an element with long-range euchromatin inducing properties. It still remains unclear as to what contains RDN1 silencing. It is possible that the cell critically monitors the level of Sir2p in order to maintain boundaries of silencing at the rDNA locus. Telomeres also nucleate the formation of silenced domain which spreads along the subtelomeric region upto ~ 2Kb. The key players in the formation of telomeric heterochromation are the Sir proteins, Sir2p, Sir3p and Sir4p, Rap1p, yKu complex and ORC. Protein-protein interactions between the telosome and the subtelomeric repeat bound silencing proteins create a domain of core heterochromatin that spreads in the adjacent sequences. While Sir2p deacetylates H4K16, Sir3p interacts with the hypoacetylated histone tails and helps in the spreading of the repressive chromatin structure. As a result telomere proximal genes are silent whereas the ones further away are expressed. There is a gradient of acetylation of histone H4, with the hypoacetylated histones at the telomeric ends and the hyperacetylated ones distant from the telomere. Recently it has been shown that this gradient is maintained by the concerted and antagonistic actions of Sir2p and Sas2p. In a sas2Δ strain Sir3p spreads to ~15 kb in the subtelomeric regions and there is increase in the levels of hypoacetylated histones. Though the molecular mechanism by which telomeric silencing is restrained is beginning to be understood, it remains unanswered whether there are any cis-acting sequences, capable of recruiting euchromatin-inducing factors such as Sas2p, near the telomeres. We have identified a RNA polymerase II driven gene, AAD3, in the subtelomeric region of chromosome III that has robust anti-silencing activity. Deletion mapping revealed that only 0.381 kb in the 5′ portion of the gene (excluding the promoter) is sufficient for barrier activity and that this property is orientation-independent (henceforth referred to as TEL-B). The barrier acivity of TEL-B depends strongly on Sas2p and Esa1p but not on Gcn5p and Sas3p, and is independent of cohesin. Previous investigations have shown that acetylation of H4K16 by Sas2p at subtelomeric regions of chromosome VI leads to deposition of HTZ1 in the nucleosome and its subsequent acetylation by Esa1p of NuA4. All these events together are required to contain the onslaught of telomeric core heterochromatin on neighbouring active regions. Since barrier activity of TEL-B depends on Sas2p and Esa1p, it is possible that TEL-B has the potential to act as a bona fide barrier in situ in its endogenous context. Our hypothesis is further cemented by the observation that there is a physical association between Sas2p, the molecule at the top of the entire cascade of events, with TEL-B by yeast one hybrid analysis. Further experiments will shed light on the role of this sequence in its natural location. In summary, I have identified and characterized two different barrier sequences in S. cerevisiae. Not many barriers are known in budding yeast and there is extensive ongoing research dedicated to understand the mechanism(s) of barrier function. In chapter I of my thesis I present a review of current literature regarding silencing barriers in yeast and other systems. In chapter II I have outlined a detailed characterization of a tDNA barrier element, tRNAGln, present near the silenced rDNA array on chromosome XII. My work addresses the various models for barrier activity and their applicability to the tRNAGln barrier. I have also attempted to understand the role of this tDNA in its natural location on the chromosome with respect to limitation of RDN1 silencing. In chapter III I have described an intensive study of a RNA polymerase II transcribed gene, AAD3, present near the right telomere of chromosome III, which acts as a robust barrier to silencing. I have attempted to answer which mechanism(s) is/are operational at this sequence so as to endow it with barrier potential. My studies with the two barrier elements highlight novel trans-acting factors required for barrier function, differential and selective requirements of certain factors for different barriers, and provide a mechanistic view of the boundary activity of these sequences.

Submitted by (ana.araujo@ufrpe.br) on 2016-08-10T13:59:35Z No. of bitstreams: 1 Rita de Cassia de Lima Idalino.pdf: 2600123 bytes, checksum: 41f878b68e3437742821d874a6955502 (MD5) / Made available in DSpace on 2016-08-10T13:59:35Z (GMT). No. of bitstreams: 1 Rita de Cassia de Lima Idalino.pdf: 2600123 bytes, checksum: 41f878b68e3437742821d874a6955502 (MD5) Previous issue date: 2010-12-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Given the large amount of data that is generated in the field of molecular genetics, is of paramount importance that techniques which allow the organization and interpretation of such data be developed and widely disseminated. Initially, we carried out a composition analysis of three gene sequences of the species: ox (Bos taurus), goat (Capra hircus), and sheep (Ovis aries), then we applied alignment techniques for identification of similarities between them. Subsequently, we used the Markov Chain theory with hidden states, i.e. Hidden Markov Models (HMMs, hereafter), in the application of discrimination problem of homogeneous regions in DNA sequences. We used the Viterbi algorithm as an auxiliary tool to obtain homogeneous regions, and then the Baum-Eelch algorithm in order to maximize the probability of a sequence of observations. We analyzed portions of HSP70.1 and NRAMP-1 genes for three different species. / Diante da grande massa de dados que é gerada na área da genética molecular, é de suma importância que técnicas que possibilitem a organização e interpretação desses dados sejam desenvolvidas e amplamente divulgadas. Inicialmente, neste trabalho, foi realizada uma análise da composição de três sequências genéticas, das espécies Bovina (Bos taurus), Caprina (Capra hircus) e Ovina (Ovis aries), em seguida aplicamos técnicas de alinhamentos para identificação de similaridades entre estas. Posteriormente, utilizamos a teoria das cadeias de Markov com estados ocultos, HMM’s (Hidden Markov Models), na aplicação do problema de discriminação de regiões homogêneas em sequências de DNA. Utilizamos o algoritmo de Viterbi como uma ferramenta auxiliar para obtenção de regiões homogêneas e em seguida o algoritmo Baum-Welch para maximizar as probabilidades de uma sequência de observações. Foram analisados trechos dos genes HSP70.1 e NRAMP-1 para três espécies diferentes.

The cell wall provides a complex and rigid structure to the plant for support, protection from environmental factors, and transport. It is mainly composed of polysaccharides, proteins, and lignin. Arabinoxylan (AX), pectin (P), and cellulose (C) are the main components of cereal cell walls and are particularly concentrated in the bran portion of the grain. Cereal arabinoxylans create networks in plant cell walls in which other cell wall polysaccharides are imbedded forming complex matrices. These networks give an insolubility profile to plant cell wall. A previous study in our lab showed that soluble crosslinked arabinoxylan with relatively high residual ferulic acid from corn bran provided advantageous <i>in vitro </i>human fecal fermentation products and promoted butyrogenic gut bacteria. In the present work, arabinoxylan was isolated from corn bran with a mild sodium hydroxide concentration to keep most of its ferulic acid content. Highly ferulated corn bran arabinoxylan was crosslinked to create an insoluble network to mimic the cereal grain cell wall matrices. Firstly, arabinoxylan film (Cax-F), pectin film (P-F), the film produced by embedding pectin into arabinoxylan networks (CaxP-F), and cellulose embedding arabinoxylan matrices (CaxC-F), and embedding the mixture of cellulose and pectin into arabinoxylan networks (CaxCP-F) were fabricated into simulated plant cell wall materials. Water solubility of films in terms of monosaccharide content was examined and revealed that Cax-F was insoluble, and P-F was partially insoluble, and nanosized pectin and cellulose were partially entrapped inside the crosslinked-arabinoxylan matrices. In a further study, these films were used in an <i>in vitro </i>human fecal fermentation assay to understand how gut microbiota access and utilize the different simulated plant cell walls to highlight the role of each plant cell wall component during colonic fermentation. <i>In vitro </i>fecal samples, obtained from three healthy donors were used to ferment the films (Cax-F, P-F, CaxP-F, CaxC-F, and CaxCP-F) and controls (free form of cell wall components -Cax, P and C). The fabricated films that were compositionally similar to cell walls were fermented more slowly than the free polysaccharides (Cax and P). Besides, CaxP-F produced the highest short chain fatty acids (SCFA) amount among the films after 24 hour <i>in vitro </i>fecal fermentation. Regarding specific SCFA, butyrate molar ratio of all films was significantly higher than the free, soluble Cax and P. 16S rRNA gene sequencing explained the differences of the butyrate proportion derived from specific butyrogenic bacteria. Particularly, some bacteria, especially in a butyrogenic genera from Clostridium cluster XIVa, were increased in arabinoxylan films forms compared to the native free arabinoxylan polysaccharide. However, no changes were observed between P and P-F in terms of both end products (SCFA) and microbiota compositions. Moreover, CaxP-F promoted the butyrogenic bacteria in fecal samples compared with pectin alone, arabinoxylan alone, and the arabinoxylan film. Differences in matrix insolubility of the film, which was high for the covalently linked arabinoxylan films, but low for the non-covalent ionic-linked pectin film, appears to play an important role in targeting Clostridial bacterial groups. Overall, the cell wall-like films were useful to understand which bacteria degrade them related to their physical form and location of the fiber polymers. This study showed how fabricated model plant cell wall films influence specificity and competitiveness of some gut bacteria and suggest that fabricated materials using natural fibers might be used for targeted support of certain gut bacteria and bacterial groups.